The invention relates to a process for the separation of enantiomers and to reagents based on an enantiopure amino acid which can be used in the separation of enantiomers.
The separation of enantiomers is a matter of great, importance in the pharmaceutical, chemical and biotechnology industries. This is because the two enantiomers of a chemical substance with an identical composition can have radically different biological activities. It is thus desirable to have available separation reagents and techniques which make it possible to separate the enantiomers and to analyse the enantiomeric purity of pharmaceutical, chemical and biotechnology products.
An article by Marfey, P., (Carlsberg Res. Comm., 49, 1984, 591-596) describes a process for the separation of enantiomers by RP-HPLC. According to this known process, 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide is used as reagent for the derivatization of amino acids. Other similar processes are also known. However, the process and reagent which are described by Marfey and the other processes and reagents exhibit numerous disadvantages.
The derivative obtained in the reaction of the amino acid with the reagent has to be isolated by successive neutralization, drying, redissolution and filtration operations. These operations take a great deal of time and are therefore not very advantageous in an industrial application. Furthermore, there is a risk, in cases of analytical applications, of errors in the analytical results caused by differing solubility of the diastereomeric derivatives in the redissolution solvent. When quantitative analyses are carried out using UV spectrometry, difficulties due to differences in absorption coefficient of the diastereomeric derivatives are encountered in the known process. Finally, the high cost of the reagent renders it desirable to find alternatives.
The invention is targeted at overcoming these problems.
The invention consequently relates to a process for the separation of enantiomers comprising at least one free functional group, in which
(a) a mixture comprising the enantiomers is reacted in basic medium with a reagent based on an enantiopure amino acid, in which reagent at least one amino group of the amino acid carries an activating group, in order to form an active precursor of an isocyanate group, and in which reagent at least one carboxyl group of the amino acid is substituted, and
(b) the mixture of diastereomers obtained is subjected to a separation operation.
It has been found, surprisingly, that the process according to the invention makes it possible to obtain good results with regard to the separation of enantiomers comprising at least one free functional group, in particular in quantitative analytical applications. The process according to the invention makes possible rapid derivatization and rapid separation of enantiomers under flexible and economical conditions.
The invention also relates to a reagent based on an enantiopure amino acid in which at least one amino group of the amino acid carries an activating group in order to form an active precursor of an, isocyanate group and in which at least one carboxyl group of the amino acid is substituted.
The term xe2x80x9camino acidxe2x80x9d is understood to denote, for the purposes of the present invention, any compound comprising at least one NH2 group and at least one carboxyl group. The amino acids used in the present invention are chiral amino acids comprising at least one asymmetric carbon. Use may be made of any chiral amino acid well known in itself of natural or synthetic origin.
Examples of reagents according to the invention are based, for example, on the following natural amino acids: alanine, valine, norvaline, leucine, norleucine, isoleucine, serine, isoserine, homoserine, threonine, allothreonine, methionine, ethionine, glutamic acid, aspartic acid, asparagine, cysteine, cystine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, ornithine, glutamine and citrulline.
Unnatural enantiomers can also be used.
Examples of amino acids of synthetic origin which can bemused as basis for the reagent according to the invention comprise, for example, the following amino acids: (1-naphthyl)alanine, (2-naphthyl)alanine, homophenylalanine, (4-chlorophenyl)alanine, (4-fluoro-phenyl)alanine, (3-pyridyl)alanine, phenylglycine, diaminopimelic acid (2,6-diaminoheptane-1,7-dioic acid), 2-aminobutyric acid, 2-aminotetralin-2-carboxylic acid, erythro-xcex2-methylphenylalanine, threo-xcex2-methylphenylalanine, (2-methoxyphenyl)alanine, 1-amino-5-hydroxyindan-2-carboxylic acid, 2-amino-heptane-1,7-dioic acid, (2,6-dimethyl-4-hydroxyphenyl)-alanine, erythro-xcex2-methyltyrosine or threo-xcex2-methyl-tyrosine.
The term xe2x80x9cenantiopure amino acidxe2x80x9d is understood to denote a chiral amino acid composed essentially of one enantiomer. The enantiomeric excess (ee) is defined: ee (%)=100(x1xe2x88x92x2)/(x1+x2) with x1 greater than x2; x1 and x2 represent the content of enantiomer 1 or 2 respectively in the mixture.
Use is generally made of an enantiopure amino acid with an enantiomeric excess of greater than or equal to 99%. Preference is given to an enantiopure amino acid with an enantiomeric excess of greater than or equal to 99.5%. In a particularly preferred way, use is made of an enantiopure amino acid with an enantiomeric excess of greater than or equal to 99.9%.
Any enantiopure amino acid can be used as basis for the reagent according to the invention. The enantiopure amino acid is preferably selected from the abovenamed amino acids of natural or synthetic origin. Amino acids comprising at least one aromatic nucleus, such as, for example, phenylalanine or its derivatives, are particularly well suited as enantiopure amino acid. In a particularly preferred way, the enantiopure amino acid is selected from phenylalanine, (1-naphthyl)-alanine, (2-naphthyl)alanine or xcex1- or xcex2-tryptophan ((2-indolyl)alanine or (3-indolyl)alanine), which are optionally substituted.
In the reagent according to the invention, at least one amino group of the enantiopure amino acid carries an activating group in order to form an active precursor of an isocyanate group.
The term xe2x80x9cactive precursor of an isocyanate groupxe2x80x9d is understood to denote any precursor which, when it is employed in a solvent which can be used in the process according to the invention with 1 equivalent of phenylalanine in the presence of 1 equivalent of base, reacts at a temperature of less than or equal to 35xc2x0 C. essentially completely in a period of time of less than or equal to 30 min to form the corresponding urea. The reactive precursor preferably releases the isocyanate group at a temperature of less than or equal to 30xc2x0 C. in a period of time of less than or equal to 15 min. In a very particular preferred way, the reactive precursor releases the isocyanate group at room temperature in a period of time of less than or equal to 10 min. Test conditions which can be used to determine the active precursor are described, for example, in Example 3 below.
The activating group is generally composed of a carbonyl derivative bonded to ant electronegative substituent. Use may: be made, for example, as activating group, of an aryloxycarbonyl, heteroaryloxycarbonyl, 1,3-imidazolyl-N-carbonyl or 1,2,4-triazolyl-N-carbonyl group. The aryloxycarbonyl groups which are well suited include those which carry at least one xe2x88x92I, xe2x88x92M substituent on an aromatic nucleus. An xe2x88x92I, xe2x88x92M substituent is a group which has a negative inductive effect and negative resonance effect as defined in J. March, Advanced Organic Chemistry, 4th Ed., 1992, p. 17-19, 273-275. The xe2x88x92I, xe2x88x92M substituents include, for example, xe2x80x94NO2, xe2x80x94SO2R, xe2x80x94SO2OR, xe2x80x94NR3+ and SR2+. The substituents are preferably found at at least one of the 2, 4 or 6 positions of the aromatic nucleus or at positions analogous to the 2 or 4 positions in condensed aromatic systems. It is preferable to use an aryloxycarbonyl activating group which carries at least one nitro substituent on the aromatic nucleus. The (4-nitrophenyloxy)carbonyl group is particularly preferred.
In an alternative form, the electronegative substituent comprises an xe2x88x92I substituent (negative inductive effect) independently of the resonance effect (M) as they have been defined above. In this alternative form, the activating group is preferably an aryloxycarbonyl group carrying at least one xe2x88x92I substituent. The xe2x88x92I substituent is preferably found at the positions which were taught above for the xe2x88x92I, xe2x88x92M substituents. Examples of xe2x88x92I substituents which can be used in the reagent according to the invention are, for example, halogens. Chlorine and fluorine are well suited. Fluorine is preferred.
In an alternative form of the invention, everything else being equal, the reagent is based on an enantiopure amino acid in which at least one amino group of the amino acid carries an activating group in order to form an active precursor of an isothiocyanate group. Use may be made, for example, as activating group, of a thiocarbonyl group bonded to an electronegative group as described above. A (4-nitro-phenyloxy)thiocarbonyl or (4-fluorophenyloxy)thio-carbonyl group is preferred.
It has been found that, in this alternative form of the invention, the reaction of the reagent with the organic compound comprising a free functional group generally gives rise to the formation of derivatives comprising a thiocarbonyl group which can give a separation of enantiomers which is further improved with respect to the oxygen-comprising derivatives. The presence of sulphur in the diastereomeric derivatives further facilitates the detection of the said derivatives, in particular by UV spectrometry.
In the reagent according to the invention, at least one carboxyl group of the amino acid is substituted.
The substituent by which the carboxyl group of the amino acid is substituted generally does not comprise a free functional group capable of reacting with the active precursor. The substituents which can be used include, for example, linear or branched alkyl groups comprising from 1 to 4 carbon atoms, such as the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl group, ethers or aryl groups, the alkyl, ethyl or aryl groups optionally being functionalized by, for example, halogens, carboxylic or sulphonic esters, sulphate esters, phosphonic esters or phosphate esters. Hydrophilic, substituents which ensure good solubility of the reagent in mixtures of water with organic solvents are well suited.
The hydrophilic substituent generally ensures that a solution of the reagent in a 1:1 by volume dioxane/water mixture is homogeneous at 20xc2x0 C. when the concentration of the reagent is at least 0.5xc3x9710xe2x88x923 mol/l. The concentration is often at least 1xc3x9710xe2x88x923 mol/l. The concentration is preferably at least 0.5xc3x9710xe2x88x922 mol/l. Good hydrophilic substituents ensure that the solution is homogeneous at 20xc2x0 C. when the concentration of the reagent is at least 1xc3x9710xe2x88x922 mol/l.
It is preferable to employ a substituent which comprises at least one ether bond. Examples of substituents comprising at least one ether bond are, for example, alkyl or aryl ethers of mono-, oligo- or polyalkylene glycols, such as, for example, mono-, oligo- or polyethylene glycol or mono-, oligo- or polypropylene glycol. The 2-methoxyethyl substituent is particularly preferred.
Particularly preferred alternative forms of the reagent according to the invention comprising a hydrophilic substituent correspond to the general formula (I) 
in which Z1 and/or Z2=NO2, R1=phenyl, xcex1- or xcex2-indolyl, 1-naphthyl or 2-naphthyl, R2=Me, Et, C3-C6 alkyl or C3-C6 cycloalkyl, and x represents an integer from 1 to 5.
In an alternative form, the substituent by which the carboxyl group or the amino acid is substituted comprises at least one chromophore. The term xe2x80x9cchromophorexe2x80x9d is understood to denote a functional group which absorbs electromagnetic radiation. The absorption maximum of the chromophores is generally from 170 to 2500 nm. The absorption maximum of the chromophores is preferably from 200 to 1000 nm. Examples of chromophores which can be used are aromatic systems optionally substituted in the 2 or 4 position by an xe2x88x92I, xe2x88x92M substituent as described above. The 4-nitrobenzyl, (2-anthraquinonyl)methyl and (9-(9H-fluorenylmethyl)) groups are particularly preferred among the substituents comprising at least one chromophore.
Preferred alternative forms of the reagent according to the invention comprising at least one chromophore correspond to the general formula (II) 
in which Z1 and/or Z2=NO2, R1=phenyl, xcex1- or xcex2-indolyl, 1-naphthyl or 2-naphthyl land Y corresponds to any one of the formulae (III to V), the carbon by which Y is bonded to the oxygen of the carboxyl group of the enantiopure amino acid being marked by *.
Use may be made of a hydrophilic substituent comprising at least one chromophore.
Particularly preferred alternative forms of the reagent according to the invention comprising an active precursor of an isothiocyanate group and a hydrophilic substituent correspond to the general formula (VI) 
in which Z1 and/or Z2=NO2 or F, R1=phenyl, xcex1- or xcex2-indolyl, 1-naphthyl or 2-naphthyl, R2=Me, Et, C3-C6 alkyl or C3-C6 cycloalkyl and x represents an integer from 1 to 5.
Preferred alternative forms of the reagent according to the invention comprising an active precursor of an isothiocyanate group and at least one chromophore correspond to the general: formula (VII) 
in which Z1 and/or Z2=NO2, R1=phenyl, xcex1- or xcex2-indolyl, 1-naphthyl or 2-naphthyl and Y corresponds to any one of the formulae (III to V), the carbon by which Y is bonded to the oxygen of the carboxyl group of the enantiopure amino acid being marked by *.
When the enantiopure amino acid comprises more than one carboxyl group, it is preferable to protect all the carboxyl groups. In a particularly preferred way, all the carboxyl groups are substituted by a substituent as described above.
When free functional groups are present in the enantiopure amino acid, it is preferable to protect the said groups by techniques known in themselves.
The reagent according to the invention can be obtained from the respective enantiopure amino acid. Known methods can be used to carry out the esterification of at least one carboxyl group of the amino acid. It is possible, for example, to employ the enantiopure amino acid and the alcohol corresponding to the substituent to be introduced in an organic solvent, such as, for example, toluene or benzene, in the presence of p-toluenesulphonic acid, preferably under azeotropic esterification conditions. Amino acid ester ammonium tosylate derivatives which are well suited to the introduction of an activating group in order to form an active precursor of an isocyanate group are obtained by this synthetic route.
Mention is made, as example of the introduction of an activating group, of the reaction of an arylox ycarbonyl chloride with the xe2x80x94NH2 group, optionally converted to the ammonium derivative, of an amino acid or of an amino acid ester in basic or neutral medium in a polar organic solvent. Tertiary amine bases, such as, for example, triethylamine or pyridine, are suitable in particular as base. When the operation is carried out in neutral medium, it is preferable to employ sodium hydrogencarbonate. Thus, good results are obtained when an amino acid ester ammonium tosylate is reacted with p-nitrophenyloxycarbonyl chloride in the presence of sodium hydrogencarbonate in a polar organic solvent, such as acetonitrile.
In the process according to the invention, the reagent according to the, invention is reacted in basic medium with a mixture comprising at least enantiomers comprising at least one free functional group.
The reaction of a reagent according to the invention, obtained from the 2-methoxyethyl ester of L-phenylalanine and from 4-nitrophenyl chloroformate, with an amino acid is illustrated in a non-limiting way in Scheme 1 below. The products of this reaction are ureas comprising two amino acids, in which at least one carboxyl functional group is substituted with a substituent. 
Generally, the free functional group is an optionally monoalkylated amino group, a hydroxyl group or a thiol group. The free functional group can also be composed of an anion, such as, for example, a carbanion or an enolate. The enantiomers comprising at least one free functional group which can be separated by the process according to the invention are generally amino acids, primary or secondary amines, peptides, alcohols, hydroxy acids or thiols. The process according to the invention gives good results in separating the enantiomers of amino acids, such as, for example, the amino acids of natural or synthetic origin mentioned above.
The process according to the invention also gives good results in separating a mixture of enantiomers of imino acids. The term xe2x80x9cimino acidxe2x80x9d is understood to denote any compound comprising at least one NHR group, in which R represents an organic radical, such as, for example, an alkyl or aryl radical, and at least one carboxyl group. Such imino acids are, for example, those belonging to the group composed of proline, pipecolic acid (piperidine-2-carboxylic acid), morpholine-3-carboxylic acid, piperazine-2-carboxylic acid, 1-thia-4-azacyclohexane-3-carboxylic acid, xcex1-methylproline, cis-4-hydroxy-proline, baikaine (1,2,3,5-tetrahydropyridine-2-carboxylic acid), cis-4-hydroxypipecolic acid, trans-5-hydroxypipecolic acid, 1,2,3,4-tetrahydronorharman-1-carboxylic acid, 1,2,3,4-tetrahydro-6-hydroxy-isoquinoline-3-carboxylic acid, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, 1,2,3,4-tetrahydro-isoquinoline-1-carboxylic acid and N-methylvaline.
The process according to the invention is carried out in a solvent system in which the mixture of enantiomers and the reagent possess sufficient solubility and the free functional group possesses sufficient nucleophilicity to react with an isocyanate. Systems comprising at least one polar organic solvent and optionally water are suitable, for example, as solvent system. Polar organic solvents which can be used are, for example, aliphatic or alicyclic ethers, such as diethyl ether, tetrahydrofuran or 1,4-dioxane, as well as aliphatic esters, such as, for example, ethyl acetate, aliphatic secondary amides, such as, for example, dimethylformamide and dimethylacetamide or, for example, N-methylpyrrolidone, or acetonitrile.
Good results for organic compounds comprising a free functional group, such as an optionally monoalkylated amino group, an arylichydroxyl group or a thiol group, are obtained in a solvent system comprising a polar organic solvent and water. Dioxane is preferred as polar organic solvent. The ratio by weight of the polar organic solvent to the water in the solvent system is generally less than or equal to 99:1. The ratio is most often less than or equal to 75:25. The ratio is generally greater than or equal to 1:99. The ratio is most often greater than or equal to 25:75.
The invention also relates to a solution of the reagent according to the invention in a polar organic solvent, such as, for example, the polar organic solvents described above. The concentration of the reagent in the solution is generally at least 1xc3x9710xe2x88x923 mol/l. The concentration is preferably at least 1xc3x9710xe2x88x922 mol/l. The concentration of the reagent in the solution is generally at most 1xc3x9710xe2x88x921 mol/l. The concentration is preferably at most 6xc3x9710xe2x88x922 mol/l. In the solution according to the invention, it is preferable to use a polar organic solvent of analytical purity. If desirable, the solution according to the invention can comprise additives, such as, for example, stabilizers.
The invention also relates to the use of the solution according to the invention in an automatic device for the derivatization and separation of enantiomers of organic compounds comprising at least one free functional group. The amount of the reagent to be employed depends on the number of free functional groups in the organic compound. At least 1 molar equivalent of reagent is employed per free functional group. Generally, at most 10 molar equivalents of reagent are employed per free functional group. Most often, at least 5 molar equivalents of reagent are employed per free functional group. Preferably, at most 3 molar equivalents of reagent are employed per free functional group. In a particularly preferred way, from 1.1 to 2.5 molar equivalents of reagent are employed per free functional group.
The basic nature of the reaction mixture is generated by known methods. The operation is preferably carried out in the presence of at least one base. Tertiary amine bases, such as, for example, triethylamine or diisopropylethylamine, which comprise one basic functionality respectively, or N,N,Nxe2x80x2,Nxe2x80x2-tetramethylethylenediamine, which comprises 2 basic functionalities, are suitable in particular as base.
The amount of base to be employed depends on the amount of the reagent and on the number of basic functionalities in the base. The molar ratio of the reagent to the basic functionalities is generally at least 1. The ratio is generally at most 2. A ratio of 1 gives good results.
In the process according to the invention, the period of time during which the reagent is reacted with the mixture comprising the enantiomers is generally less than or equal to 30 min. Most often, the period of time is less than or equal to 20 min. Preferably, the period of time is less than or equal to 15 min. Good results are obtained with a period of time of greater than or equal to 15 seconds. In practice, a period of time of greater than or equal to 1 min is most often applied. A period of time of 5 to 15 min is highly suitable.
The temperature at which the reagent is reacted with the mixture comprising at least the enantiomers of an organic compound is generally has than or equal to 35xc2x0 C. The temperature is most often less than or equal to 30xc2x0 C. The temperature is generally greater than or equal to xe2x88x9220xc2x0 C. The temperature is most often greater than or equal to 0xc2x0 C. In a particularly preferred way, the temperature is room temperature, that is to say generally from 15 to 30xc2x0 C., preferably 20 to 25xc2x0 C.
In the process according to the invention, the mixture of diastereomers obtained is subjected to a separation operation. The separation operations which can be used for the separation of a mixture of diastereomers are described, for example, in E. Eliel, Stereochemistry of Organic Compounds, 1994, p. 344-381. Mention may be made, as examples, of distillation, crystallization and gas or liquid chromatography operations. Among these operations, liquid chromatography operations, such as, for example, HPLC chromatography, are preferred. In a particularly preferred way, the separation operation is RP-HPLC (reverse phase) chromatography. Information regarding HPLC chromatography is found, for example, in Rompp Chemie-Lexikon, 9th Ed., p. 1860-1861. Use may also be made of thin layer chromatography.
Eluents which can be used in a chromatography operation are known. In the case where the process according to the invention comprises RP-HPLC chromatography as separation operation, good results have been obtained with an eluent comprising acetonitrile or methanol.
In an alternative form of the process according to the invention, which is preferred, the mixture of diastereomers obtained is subjected to the separation operation without prior purification. In known methods for the separation of enantiomers, a crude mixture of diastereomers is isolated, which mixture has to be subjected to purification prior to the separation of the diastereomers. The process and the reagent according to the invention make it possible not to isolate the crude mixture of diastereomers and to carry out the separation operation without prior purification.
The process and the reagent according to the invention can be used for the preparative or analytical separation of enantiomers. The process and the reagents are well suited to the analytical separation of enantiomers. In an alternative form, the process and the reagent are used to determine the enantiomeric excess of an amino acid or of a primary or secondary amine. In another alternative form, the process and the reagent are used to determine the enantiomeric excess of a peptide.
The invention also relates to a process for the production of an enantiopure compound comprising at least one free functional group in which:
(a) a mixture comprising the enantiomers of the compound comprising at least one free functional group is subjected to the separation process according to the invention
(b) a cleavage operation is carried out on a pure diastereomer obtained by separation of the mixture of diastereomers
(c) the enantiopure compound is recovered.
Use may be made, as cleavage operation, of, for example, an operation of hydrazinolysis in a solvent, such as, for example, an alcohol.
When the process and the reagent according to the invention are used for the analytical separation of enantiomers, use is made of a detection technique known in itself for the determination of the content of enantiomers in the mixture. Optical techniques, such as, for example, UV spectrometry, visible spectrometry or fluorimetry are highly suitable as detection technique.